Rapid detection of infectious agents has become more relevant with concerns associated with detection of bioterrorist agents. Current technology for detection and quantification of clinically relevant molecules is based on monoclonal and polyclonal antibodies used for immunoassay or detection of nucleic acids using RT-PCR systems. These assays are relatively slow taking at least 3 hours to complete. A novel, rapid, immunoproteomics assay based on high affinity specific antibodies coupled with a mass spectral read out is proposed. In a series of preliminary studies we have demonstrated the ability to use a single antibody-coated antigen capture and transfer reagent (ACTR) to detect model antigens, with good sensitivity, using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF). This immunoproteomic assay can be complete in < 1 hour and follows a sequential process in which antibody is immobilized in step 1, antigen is captured in step 2 and bound antigen is analyzed by mass spectrometry in step 3. Immunoproteomic assays may potentially be faster and as sensitive as the current antibody assays and could provide evidence for bacterial or viral antigens or specific antibodies to these products in samples for an infected animal. This RO3 proposal is designed to optimize this platform technology. In the initial proof-of-concept studies, we will develop and evaluate immunoproteomic assays to measure INF gamma (Th1 cytokine) and specific anti-viral antibodies in the serum of ectromelia infected mice. Ectromelia is a surrogate of variola virus, a CDC Category A bioterrorist agent and thus, the successful immunoproteomic approach may be relevant for detection of bioterrorist agents.